Amylase mutant having high specific activity and thermal stability, gene of mutant, and applications thereof

ABSTRACT

The present invention relates to the field of agriculture biotechnology, specially relates to an amylase mutant having high specific activity and thermal stability, gene and use thereof. Said amylase mutant is obtained by performing substitution of S33A/S34E/V35H, and deletion of amino acids at the sites of 178 and 179 of the wild type amylase having amino acid sequence of SEQ ID NO:1, and having improved enzymatic activity and thermal stability than the wild type amylase.

FIELD OF THE INVENTION

The present invention relates to the field of genetic engineering,particularly to an amylase mutant having high specific activity andthermal stability, gene and application thereof.

BACKGROUND OF THE INVENTION

Amylase, also known as 1,4-α-D-glucan hydrolase (EC 3.2.1.1), is anenzyme that can hydrolyze α-1,4-glycosidic bond in starch, which hasbeen widely applied to food, medicine, feed, textile and otherindustries.

At present, the industrially produced amylase is mainly from Bacillus.The conventional methods of producing the amylase focus on obtaininghigh-yield strains with UV or the chemical mutagens, and optimizing thefermentation conditions of the strains to further improve the enzymeproduction capacity of strains. However, the fermentation process ofBacillus is relatively complex, and has the drawbacks of low yield anduneven fermentation products, which is disadvantageous to the laterprocessing technology. Therefore, the genetic engineering technology isapplied to improve amylase production. Although the heterologousexpression of amylase is realized by genetic engineering technology,it's still to resolve the problem of the high production cost caused bythe low expression level, which limits the large-scale industrialproduction and application of the amylase. Therefore, the method of theprotein engineering is applied to molecular modification of the amylaseto improve its catalytic performance.

Order of the Invention

One order of the present invention is to provide a amylase mutantobtained by mutation of site or sites of the amino acid sequence of theamylase from Bacillus amyloliquefaciens.

Another order of the present invention is to provide a gene encoding theabove mutant.

Another order of the present invention is to provide a recombinantvector comprising the gene encoding the above mutant.

Another order of the present invention is to provide a recombinant cellcomprising the gene encoding the above mutant.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides an amylase mutant which isobtained by performing substitution of S33A/S34E/V35H to acid aminosequence of the wild type amylase with amino acid sequence as shown inSEQ ID No:1, and removing amino acids of the sites of 178 and 179, or anamylase mutant having 90-99% sequence identity to the amylase obtainedby performing the substitution of S33A/S34E/V35H to the acid aminosequence of the wild type amylase with amino acid sequence as shown inSEQ ID No:1, and removing amino acids of the sites of 178 and 179,wherein said amylase mutant has 2.5-3.5 times activity of the wild typeamylase, remains more than 99% of its initial activity after 5 min'sincubation at 70° C., and remains 32% of its initial activity after 5min's incubation at 80° C.

SEQ ID No: 1: AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLSSVGITAVWTPPAYKGTSQADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGDVVMNHKAGADYTENVTAVEVNPSNRNQETSGEYNIQAWTGFNFPGRGTTYSNFKWQWFHFDGTDWDQSRSLSRIFKFRGTGKAWDWEVSSENGNYDYLMYADIDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFSFLKDWVDNARAATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGGYYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAFILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQRDYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWYDLTGNRTDKITIGSDGYATFPVNGGSVSVWVQQ*

In a preferred embodiment the amylase mutant according to the presentinvention has the amino sequence of SEQ ID NO: 2.

SEQ ID NO: 2: AATNGTMMQYFEWYVPNDGQQWNRLRTDAPYLAEHGITAVWTPPAYKGTSQADVGYGPYDLYDLGEFNQKGTVRTKYGTKGELKSAVNTLHSNGIQVYGDVVMNHKAGADYTENVTAVEVNPSNRNQETSGEYNIQAWTGFNFPGRGTTYSNFKWQWFHFDGTDWDQSRSLSRIFKFTGKAWDWEVSSENGNYDYLMYADIDYDHPDVVNEMKKWGVWYANEVGLDGYRLDAVKHIKFSFLKDWVDNARAATGKEMFTVGEYWQNDLGALNNYLAKVNYNQSLFDAPLHYNFYAASTGGGYYDMRNILNNTLVASNPTKAVTLVENHDTQPGQSLESTVQPWFKPLAYAFILTRSGGYPSVFYGDMYGTKGTTTREIPALKSKIEPLLKARKDYAYGTQRDYIDNPDVIGWTREGDSTKAKSGLATVITDGPGGSKRMYVGTSNAGEIWYDLTGNRTDKITIGSDGYATFPVNGGSVSVWVQQ*

In a yet preferred embodiment of the present invention, said amylasemutant is the mutant obtained substitution, deletion and/or insertion ofone or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9, amino acid residues ofpolypeptide of SEQ ID NO:2, and maintaining the properties of the aboveamylase mutant. For example, a common strategy is substitutions of theconservative amino acid that the amino acid residue is replaced withanother amino acid residue having a similar side chain without effect onthe properties of the enzyme. Families of amino acid residues havingsimilar side chains have been defined in the art. Furthermore, it iswell known in the art that the suitable peptide linker, signal peptide,leader peptide, terminal extensions, glutathione S-transferase (GST),maltose E binding protein, protein A, tags such as 6His or Flag, orproteolytic cleavage site for Factor Xa, thrombin or enterokinase areusually introduced into the N- or C-terminus of the recombinant proteinor within other suitable regions of the proteins, in order to constructa fusion protein, to enhance expression of recombinant protein, toobtain an recombinant protein automatically secreted outside the hostcell, or to aid in the purification of the recombinant protein.

In a further preferred embodiment, a mutant amylase has the sequenceidentity of least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,more preferably at least about 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%,98.7%, 98.8%, 98.9%, and even more preferably at least about 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more to thefull amino acid sequence of SEQ ID NO:2. Ranges and identity valuesintermediated to the above-recited values are also intended to beincluded in the present invention.

According to the prior art in the art, the mutant refers to theindividual which is obtained by mutation of the wild type protein, andhas the phenotypic characteristics different from the wild type. Aperson skilled in the art can obtain the mutant having the newproperties by replacing, inserting or deleting specific nucleotides ofthe DNA sequence under the condition of knowing the structure andfunction of the protein.

In a further preferred embodiment the present invention provides thegene encoding the above amylase mutant having high activity andthermostability. The said gene can be the molecule of DNA, cDNA, mRNA,hnRNA, or tRNA.

In a further preferred embodiment, the present invention provides a genehaving a nucleotide sequence which hybridizes to a nucleotide sequenceof SEQ ID NO: 2 under stringent conditions. As used here, the term“hybridize under stringent conditions” refers to the hybridization andcleaning conditions in which at least 90% of homologous nucleotidesequences can still be hybridized with each other. The said stringentcondition are well known to those skilled in the art and can be found incurrent protocols in molecular biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. One example of hybridization under stringent conditions ishybridization in 6×SSC at 45° C., then washing one or more times at50-65° C. in 0.2×SSc and 0.1% SDS. Those skilled in the art canunderstand that highly stringent conditions can be achieved byincreasing the hybridization temperature, for example, to 50° C., 55°C., 60° C. or 65° C.

In addition, those skilled in the art will understand that there mayexist the genetic polymorphism due to natural variation amongindividuals of a population. The gene encoding the amylase mutant of thepresent invention may have such natural variation without changing theactivity of the mutant. Therefore, the present invention also includesalleles of a gene encoding a polypeptide having an amino acid sequenceof SEQ ID No:2.

In another aspect, the present invention provides recombinant vectorcomprising the gene encoding the abovementioned amylase mutant. In apreferred embodiment of the present invention, the said recombinantvector is constructed based on the vector of pHYP16. The recombinantexpression vectors of the invention can be designed for expressingamylase in prokaryotic or eukaryotic cells. For example, amylase can beexpressed in bacterial cells such as E. coli, yeast such as Pichia orAspergillus, insect cells such as Sf9 cell or silkworm cell withbaculovirus expression vectors, or plant cell such as Arabidopsis,tobacco, corn, and so on, mediated by Agrobacterium tumefaciens. Thus,the invention relates to host cells introduced with a recombinantexpression vector of the invention. The host cells of the presentinvention may be any prokaryotic or eukaryotic cell, including but notlimited to the above host cells. Preferably, said host cell is Pichiapreferred. Pichia pastoris is methylotrophic yeast, capable ofmetabolizing methanol as its sole carbon source. This system iswell-known for its ability to express high levels of heterologousproteins. As an effective expression system, many of the gene encodingthe amylase have successfully expressed in P. pastoris. The novel geneencoding the mutant amylase of the present invention is also expressedin P. pastoris with high levels. So it will be very easy to mass-producethe polygalacturonase by fermentation in the lower cost than ever.

In a preferred embodiment, the vector DNA can be transferred intoprokaryotic or eukaryotic cells by the conventional transformation ortransfection methods. Appropriate methods for transforming ortransfecting host cells can be found in the second edition of Molecularcloning (Sambrook et al.), and other laboratory manuals.

In a preferred embodiment, the present invention provides a recombinantstrain comprising the above gene encoding the said mutant amylase.Preferably, said recombinant strain is E coli, yeast such asPichiapastoris cell, Saccharomyces cerevisiae, or Hansenulapolymorpha,Bacillus or Lactobacillus, more preferably Bacillus SCK6 cells.

In another aspect, the present invention also relates to a method ofproducing amylase having high activity and thermostability comprisingthe steps of:

(1) transforming a host cell with the DNA construct or a recombinantvector of comprising said gene encoding the above amylase mutant toobtain the recombinant host cell;

(2) cultivating the recombinant host cell to induce the expression ofamylase; and

(3) isolating and recovering said amylase

Compared with the wild-type amylase, the mutant amylase of the presentinvention has the following profiles: (1) having the increased enzymeactivity which is the 2.5-3.5 times of the wild type, such as 2.5 times,2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 3.1 times, 3.2times, 3.3 times, 3.4 times or 3.5 times; (2) having the improvedthermostability of retaining more than 99% of activity for 3 to 10 min,such as 3 min, 4 min, 5 min, 6 min, 7 min, 9 min or 10 min, at 70° C.,and retaining 30% to 35% of activity for 3 min, 4 min, 5 min, 6 min, 7min, 9 min or 10 min at 80° C.

In another aspect, the invention provides the application of the amylasemutant with high specific activity and thermal stability, wherein saidmutant amylase can be applied to food, medicine, animal feed and/ortextile industry.

Also, the invention provides the application of gene encoding the abovemutant amylase to food, medicine, animal feed and/or textile industry.

The present invention overcomes the shortcomings of the prior art andprovides a amylase mutant with high enzyme activity and excellentthermal stability, which can be applied to food, medicine, feed, textileindustry, etc. The mutant S33A/S34E/V35H/Δ R178/G179 of the presentinvention has the enzyme activity increasing to 17067.57 U/Mg from5553.53 U/mg of wild type; still retains more than 99% of the enzymeactivity after being treated at 70° C. for 5 min, compared withremaining 10% for the wild type; and retains 32% of the enzyme activityafter being treated at 80° C. for 5 min, compared with almost losing allof enzyme activity for the wild type. Therefore, the amylase mutantamylase of the present invention can meet the requirements ofapplication in food, medicine, feed and textile industry, and has a verybroad application prospect.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 shows the enzymatic properties of the wild-type amylase;

FIG. 2 shows the comparison of thermal stability of the amylase mutantwith that of the wild-type amylase;

FIG. 3 shows the SDS-PAGE electrophoresis results of amylase expressedin Bacillus subtilis sck6.

EMBODIMENT

The present invention is further illustrated with reference to thefollowing examples and the appended drawings, which should by no meansbe construed as limitations of the present invention.

Test Materials and Reagents

1. Strains and vectors: host: Bacillus subtilis SCK6; and vectorpHYP16-BKAMY

2. Enzymes and other biochemical reagents: restriction endonucleases(Fermentas); and ligase (Promaga).

3. Medium: LB medium; starch medium

Suitable biology laboratory methods not particularly mentioned in theexamples as below can be found in Sambrook, et al. (Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other kitlaboratory manuals.

Example 1 Site Directed Mutagenesis of the Gene Encoding the Amylase

The optimized mutation site wasS33A/S34E/V35H/ΔR178/Δ G179. The mutationsite was introduced with the primers of table 1 by Site-directedMutagenesis Kit followed by sequencing to obtain the mutant gene.

TABLE 1 Primers SEQ Length ID NO. Primers Sequence (5′→ 3′)^(a) (bp) 3Bkamy-F ttacaaaaacatcagccgtaggatccg 50 ccgcaacgaacggaacaatgatg 4 Bkamy-Rgggacgtcgacttagtggtggtggtgg 54 tggtgctgctgaacccacactgagacg 5 pHYP16-Fgggttcagcagcaccaccaccaccacc 51 actaagtcgacgtccccggggcag 6 pHYP16-Rattgttccgttcgttgcggcggatcct 50 acggctgatgtttttgtaatcgg

Sequencing results showed that the nucleotide sequence amplified bymutation had 1458 bp, comprising the encoding area of 1455 bp encodingthe amino acid sequence of SEQ ID No: 2 having 485 amino acid residues.The protein with the amino acid sequence of SEQ ID No: 2 was named asamylase mutant BKAMYA.

Example 2 Preparation of Amylase Mutant BKAMYA

1. Preparation of Recombinant Plasmid pHYP16-BKAYA

Firstly, the vector and the target fragment were amplified by POE-PCRfollowed by being recovered and mixed in a proper proportion, and thenadded into the PCR system to construct the recombinant plasmidcontaining the said amylase gene. Mutation of nucleotides was introducedby Site Mutation kit using the wild type plasmid as the temple. Then,the obtained recombinant plasmid was sequenced to test the validity ofpurpose sequence. The recombinant plasmid inserting the exogenous genewas named aspHYP16-BKAMYA.

2. Preparation of the Recombinant Strain SCK6/BKAMYA

The above recombinant plasmid pHYP16-BKAMYA was transformed intoBacillus spk6 cells to obtain the recombinant strain SCK6/BKAMYA.

3. Preparation of the Amylase Mutant

The above recombinant strainSCK6/BKAMYA was inoculated into 50 mL ofmedium in 100 mL of flask and cultured on a shaker operating at 220 rpmat 37° C. and for 24 h. Then, the cultured medium was transferred to 200mL medium in a 1 L of flask to culture at 37° C. and 220 RPM again. Thesupernatant was collected by centrifugation to purify the amylase mutantBKAMYA by affinity chromatography for analyzing activity.

Example 3 Analysis and Comparison of the Amylase Mutant BKAMYA and WildType Amylase

1. Analysis and Comparison of the Enzymatic Activity

The enzymatic activity of amylase was determined with UVspectrophotometer by the steps of performing the enzymatic reaction atthe certain temperature and pH for 20 min, wherein 1 mL of saidenzymatic reaction system included 100 μL of appropriate diluted enzymesolution and 900 μL of substrate, measuring the absorbance at 540 nm andcalculating the enzymatic activity. A unit of enzymatic activity (U) isdefined as the amount of enzyme to produce 1 μmol glucose per unit timeunder given conditions.

The purified amylase mutant prepared in example 2 and the wild typeamylase were performing the enzymatic reaction at pH 7.0 and 55° C. todetermine their enzymatic activity.

As showed in FIG. 1, the enzymatic activity of wild type is 5553.53U/Mg, and that of amylase mutant is 17067.57 U/mg.

2. Analysis and Comparison of Thermal Stability

The thermal stability of both the purified amylase mutant prepared inexample 2 and the wild type was determined by treating at 60° C. for 10min, 30 min and 60 min respectively or 70° C. for 2 min, 5 min, and 10min respectively, in 0.1 mol/L of citric acid-disodium hydrogenphosphate buffer (pH 7.0), and their retained enzymatic activity at 55°C. was determined.

As showed in FIG. 2, the mutant S33A/S34E/V35H/ΔR178/G179 retained morethan 99% of enzymatic activity, but the wild type amylase retained about10% of enzymatic activity, after being treated at 70° C. for 5 min; and,the mutant retained 32% of enzymatic activity, but the wild type almostlost enzymatic activity after being treated at 80° C. for 5 min. And,the specific activity of mutant was increased to 17067.57 U/mg comparedwith that of the wild type amylase.

1. An amylase mutant, (1) being obtained by substituting S33A/S34E/V35H,and deleting the amino acids at the sites of 178 and 179 of the wildtype amylase having amino acid sequence of SEQ ID NO:1, and havingimproved enzymatic activity and thermal stability than those of the wildtype amylase, or having 99% sequence identity to the polypeptideobtained by substituting S33A/S34E/V35H, and deleting the amino acids atthe sites of 178 and 179 of the wild type amylase having amino acidsequence of SEQ ID NO:1; and (2) having the 2.5-3.5 times enzymaticactivity of the wild type, retaining more than 99% and 30%˜35% of theenzymatic activity after being treated at 70° C. and 80° C.respectively.
 2. The amylase mutant according to claim 1, wherein saidamylase mutant retains more than 99% of enzymatic active after beingtreated at 70° C. for 3 to 10 min.
 3. The amylase mutant according toclaim 1, wherein said amylase mutant retains 32% of enzymatic activeafter being treated at 80° C. for 3 to 10 min.
 4. A gene encoding theamylase mutant of claim
 1. 5. A recombinant vector of comprising thegene of claim
 4. 6. A recombinant cell comprising the gene of claim 4.7. A method of preparing amylase having high enzymatic activity andthermal stability, comprising the steps of (1) constructing arecombinant vector comprising the gene of claim 2; (2) transforming thehost cell with the recombinant vector obtained by the step (1); and (3)culturing said host cell and isolating said amylase.
 8. A use of theamylase mutant of claim
 1. 9. A use of the amylase mutant of claim 1 tofeed, medicine, food or textile fields.
 10. A use of the gene of claim4.
 11. A use of the gene of claim 4 to feed, medicine, food or textilefields.